Authentication of gadoids from highly processed products susceptible to include species mixtures by means of DNA sequencing methods

Economic importance of gadoids such as fishing resource, and their conservation status, necessitates the development of techniques that allow unequivocal authentication of products made from them. Amplification of a fragment of mitochondrial cytochrome b (cyt b) marker and subsequent phylogenetic analysis were carried out to authenticate these products and assure their correct labeling. Also, SNP analysis that allows detection of species mixtures of Gadus genus was developed. For this, two fragments of the cyt b gene were amplified and sequenced, one of 464 bp and another internal fragment to this of 263 bp to allow the authentication of gadoid species in highly processed products. Obtained sequences were aligned and analyzed in order to assess the presence of informative variable positions and a maximum of 14 SNP were identified and selected. These allow detection and identification of species mixtures belonging to this genus. The developed methodologies were validated and applied to 25 commercial samples. The main novelty of this work lies in the fact that is the only work that allows the detection of species mixtures of the Gadus genus and is the only one that allows the authentication of highly processed products up to date. Furthermore, this methodology allows identifying more of 15 species of gadoids and can be applied to all kinds of seafood products. Therefore, this molecular tool can be applied in questions related to check the fulfillment of labeling regulations for seafood products, verify the correct traceability in commercial trade and for fisheries control.     

Development of a method for the identification of scombroid and common substitute species in seafood products by FINS

In the present work, a method for the authentication of scombroid products was developed, by means of FINS (Forensically Informative Nucleotide Sequencing) technique (Polymerase Chain Reaction (PCR) followed by phylogenetic analysis). The methodology developed allows the identification of most important scombroid species using the mitochondrial cytochrome b as molecular marker. Due to the different commercial value of the species belonging to this family, substitutions between species in seafood products can take place.     

Species authentication of octopus,cuttlefish, bobtail and bottle squids (families Octopodidae,Sepiidae and Sepiolidae) by FINS methodology in seafoods

In the present work a molecular a method for the authentication of cephalopods products was developed, which allows the genetic identification of about 30 species belonging to the families Octopodidae, Sepiidae and Sepiolidae. This molecular system is based on the phylogenetic analysis of DNA sequences. The molecular marker studied was the cytochrome b gene (cyt b), that was amplified by PCR and subsequently sequenced. The developed methodology was validated and further applied to 20 commercial samples, detecting 6 that were incorrectly labelled (30%). Therefore, this molecular tool could be applied in questions related to correct labelling, traceability, and import control of products containing the taxonomic groups studied.     

Development of a rapid and simple molecular identification methodology for true sardines (Sardina pilchardus) and false sardines (Sardinella aurita) based on the real-time PCR technique

Sardines are a small pelagic species belonging to the Clupleidae family and have high commercial value because of the properties of their meat. One of the most valuable fish in this family is Sardina pilchardus, which in addition to its high commercial value is the one that should be labeled on canned sardines, the other species being labeled as sardines X. In this study, a fast real-time polymerase chain reaction (PCR) assay based on TaqMan probe technology, which amplifies a fragment of the internal transcribed spacers (ITS1 and ITS2) and the 5.8 S rRNA region, was developed for the authentication of two commercially important species of sardines, S. pilchardus and S. aurita. This methodology can be applied to a variety of products, including those that have been subjected to intensive processing treatments such as canning. The present methodology was validated and subsequently applied to 50 commercial samples in order to determine whether correct labeling is being carried out in the market. This assay combines the high specificity, sensitivity, and rapidity of fast real-time PCR with the advantages of using two different probes in the same reaction, making it a powerful tool for verifying the correct application of food labeling regulations.     

FINS methodology to identification of sardines and related species in canned products and detection of mixture by means of SNP analysis systems

The sardines are a resource of great importance in the artisanal and industrial fisheries worldwide. Such sardines and sardine-type products are included many species of small pelagic species, all belonging to the Clupeidae family. Within this family, highlights the European sardine (Sardina pilchardus), which by its organoleptic characteristics this species has gained an extraordinary commercial significance. In this work, an amplification of a fragment of mitochondrial cytochrome b marker and subsequent phylogenetic analysis (FINS: Forensically Informative Nucleotide Sequencing) were carried out to assure the correct labeling of sardine and sardine-type products. On the other hand, a single nucleotide polymorphism (SNP) analysis that allows detection of mixture of S. pilchardus and S. aurita in canned products was developed. After the application of this methodology to more than 80 available commercial samples can conclude that in more of 15% of the products analyzed, the name of the species displayed in the label was not in agreement with the identified species, also a one of these included in mixture of species. The main novelty of this work lies in the fact that have been included a long number of different species of sardines, many of which were not included in similar works up to date. Furthermore, this methodology allows identifying over 20 species of sardines and can be applied to all kinds of processed products, including those who have been subjected to intensive processing treatments, such as canned foods. Therefore, this molecular tool can be applied in questions related to correct labeling, traceability, fishery regulations, and commercial trade control of sardines and sardine-type products.     

Duplex real-time PCR for authentication of anglerfish species

A duplex real-time PCR assay based on TaqMan probe technology was developed for the authentication of two commercially important anglerfish species (Lophius budegassa and L. piscatorius). This technique uses the cytochrome oxidase subunit I and allows the unambiguous identification of this species. It is notable for its simplicity, rapidity, highest potential for automation and minor risk of contamination. The TaqMan real-time PCR is currently the most suitable method for screening, allowing the detection of fraudulent or unintentional mislabeling of these species. The method can be applied to all kind of products as fresh, frozen, and precooked products. The developed methodology, which uses two specific primer–probe sets, has been validated and subsequently applied to 40 commercial samples labeled as any anglerfish species in order to determinate whether the species used for their manufacturing correspond to these species. The methodology herein developed is appropriate to clarify questions related with the correct labeling of commercial products, traceability in commercial trade, and fisheries control.     

Authentication of the most important species of rockfish by means of fins

In the present work, a method for the authentication of more of 100 scorpaenids species has been developed by means of forensically informative nucleotide sequencing technique, which is based on a PCR followed by a phylogenetic analysis. Due to the different commercial value of the species belonging to this taxonomic group, substitutions between species in seafood products can take place. Two different methodological strategies are proposed, allowing the analysis of fresh, frozen, or highly processed products with total reliability. These analytical systems have been validated and subsequently applied to 25 commercial samples. Therefore, this technique can be used as a routine method to avoid the mislabeling in the marketing of scorpaenid species and it is also suitable to assess the correct seafood traceability of these products.     

Species identification and animal authentication in meat products: a review

Meat species identification and animal authentication in meat products is a significant subject, attention to which would contribute to fair-trade, and would enable consumers to make informed choices. Analytical methods are often based on protein or DNA measurements. Methods based on protein fractions include electrophoretic, chromatographic and immunological techniques and are often not suitable for compound food products, nor are they sensitive in processed products to differentiate closely related meat species. Advances in DNA technology have led to the rapid development of alternative approaches to species identification. Recently, application of polymerase chain reaction in food analysis has increased in the light of their simplicity, specificity and sensitivity. This review discusses a wide range of analytical methods with a focus on their ability to quantify meat and authentication of meat products.     

Biochip Technology for the Detection of Animal Species in Meat Products

The verification of declared components in meat products is an essential task of food control agencies worldwide. To date, the ELISA and species-specific polymerase chain reaction (PCR) are two commonly applied analytical tools employed by many authorized food control laboratories. These trusted methods however do not allow the simultaneous detection of all the animal species present in a meat sample. Additionally, detection of undeclared components resulting from inadvertent contamination or deliberate adulteration of the meat products requires additional processing of the samples, resulting in increased expenditure. The use of DNA biochip analysis that allows simultaneous processing of many meat products, while concomitantly generating results for the detection of all animal species present in the meat products is thus highly desirable. In this work, two commercially available animal chip detection systems (CarnoCheck Test Kit and MEAT^species LCD Array) are compared in terms of sensitivity, robustness, reproducibility, and ease of handling. The two animal species differentiation biochip methods compared well in efficiency and could simultaneously detect from eight to 14 animal species in the meat products. Detection limits were found to be in the range of 0.1% to 0.5% in meat admixtures, with good reproducibility of results. More than 70 commercially available meat samples were analyzed in this work, with the results validated against traditional PCR methodology. Both biochip methods performed well and could be implemented for routine use in any food control agency.     

Rapid method for controlling the correct labeling of products containing European squid (<Emphasis Type="Italic">Loligo vulgaris</Emphasis>) by fast real-time PCR

The squids are a group of cephalopods widely distributed and with high commercial value. In the present work, a fast real-time PCR was developed for the authentication of the European squid (Loligo vulgaris). This method is based on a specific primer/probe set that amplifies a fragment of the Internal Transcribed Spacer 1 (ITS 1) ribosomal DNA region. This technique is notable for its conceptual and practical simplicity, together with its combination of speed, sensitivity and specificity in a homogeneous assay. To all this must be added the time savings produced by the fast real-time PCR due to shorter runs. The presented methodology was validated to check how the degree of food processing affects the applicability of this technique and therefore the detection of L. vulgaris. It was demonstrated that the technique can be applied to all kinds of processed products. The commercial denomination of some cephalopods, including European squids, is an important issue due to the legal gaps, since the same species has different commercial name depending on the format in the market. The methodology herein developed was applied to 42 commercial samples to evaluate the situation regarding the labeling of products made from these species. Moreover, the method can be applied to all kinds of products regardless of the degree of processing.     

A fast and accurate method for controlling the correct labeling of products containing buffalo meat using High Resolution Melting (HRM) analysis

The substitution of high priced meat with low cost ones and the fraudulent labeling of meat products make the identification and traceability of meat species and their processed products in the food chain important. A polymerase chain reaction followed by a High Resolution Melting (HRM) analysis was developed for species specific detection of buffalo; it was applied in six commercial meat products. A pair of specific 12S and universal 18S rRNA primers were employed and yielded DNA fragments of 220 bp and 77 bp, respectively. All tested products were found to contain buffalo meat and presented melting curves with at least two visible inflection points derived from the amplicons of the 12S specific and 18S universal primers. The presence of buffalo meat in meat products and the adulteration of buffalo products with unknown species were established down to a level of 0.1%. HRM was proven to be a fast and accurate technique for authentication testing of meat products.     

Traceability in the meat industry – the farm to plate continuum

Traceability of meat and meat products is a major issue in the meat industry with the two main drivers being food safety/risk management and authentication. Increasingly, the world marketplace is indicating that traceability systems for food products derived from individual animals (e.g. steak, chops etc.) is required now or will be required in the near future. Traceability requirements for compound products, such as ground beef, are usually less strict and are frequently limited to date and place of manufacture. As global competition increases it is imperative that technologies are available that protect against, and deter, fraudulent labelling of inferior product. Traceability offers more than marketing advantages. It can be applied at every stage of the meat production continuum and can be just as valuable to farmers and processors as it is to marketers and consumers.     

A SYBR Green real-time PCR assay to detect and quantify pork meat in processed poultry meat products

Species identification in meat products has grown in interest in recent years since these foodstuffs are susceptible targets for fraudulent labelling. In this work, a real-time PCR approach based on SYBR Green dye was proposed for the quantitative detection of pork meat in processed meat products. For the development of the method, binary meat mixtures containing known amounts of pork meat in poultry meat were used to obtain a normalised calibration model from 0.1 to 25% with high linear correlation and PCR efficiency. The method revealed high specificity by melting curve analysis, being successfully validated through its application to blind meat mixtures, which confirmed its adequacy for pork meat determination. The fully applicability of the method was further demonstrated in commercial meat products, allowing verification of labelling compliance and identification of meat species in processed foods.     

微滴式数字聚合酶链式反应精准定量检测羊肉中掺杂猪肉

以羊和猪的单拷贝持家基因DNA复制蛋白A1为靶基因设计合成了适用于微滴式数字聚合酶链式反应的特异性引物和探针,通过理论推导获得了单位质量两种肉基因拷贝数之比的固定值,并进行了验证,据此将样品中羊肉和猪肉的拷贝数转换为相对质量分数,从而建立了羊肉中掺杂猪肉的精准定量检测方法。该方法可以很好地应用于羊肉中掺杂猪肉的含量检测,猪肉的最低定量限为1%,在5%~80%范围内绝对误差小于±1.30%,相对误差小于±10%,定量结果准确、重复性高,可以为肉类掺假的监管工作提供有力的技术参考。     

Real-time PCR method applied to seafood products for authentication of European sole (Solea solea) and differentiation of common substitute species

Judged by quality and taste, the European sole (Solea solea) is considered one of the finest flatfish and is, thus, of considerable commercial value. In the present work, a specific fast real-time PCR was developed for the authentication of S. solea, i.e. to distinguish it from other related species and avoid substitution of this species, either deliberately or unintentionally. The method is based on a species-specific set of primers and MGB Taqman probe which amplifies a 116-bp fragment of the internal transcribed spacer 1 (ITS 1) ribosomal DNA region. This assay combines the high specificity and sensitivity of real-time PCR with the rapidity of the fast mode, allowing the detection of S. solea in a short period of time. The present methodology was validated for application to all types of manufactured products for the presence of S. solea, with successful results. Subsequently, the method was applied to 40 commercial samples to determine whether correct labeling had been employed in the market. It was demonstrated that the assay is a useful tool in monitoring and verifying food labeling regulations.     

DNA条形码技术在肉及其制品中的应用

DNA条形码技术作为一种新的分子生物学检测技术在鉴定和区分各物种和物种间亲缘关系方面得到了广泛应用,该技术能实现对肉的快速、准确检测。DNA条形码已成为生物学领域发展最迅速的一种技术,该技术基于广泛的物种基因数据库信息,在肉品研究中具有良好的应用前景。本文简述DNA条形码技术的基本原理,并基于DNA水平上与其他相关技术进行比较,综述其在物种鉴定、肉品质量安全、商业欺诈等方面的应用,并对该技术在今后肉品科学研究中的应用进行展望。      

Canine-Specific PCR Assay Targeting Cytochrome b Gene for the Detection of Dog Meat Adulteration in Commercial Frankfurters

This report described a cytochrome b (cytb)-based polymerase chain reaction (PCR) assay for the detection of canine tissues in commercial frankfurters. Discriminating detection of canine derivatives in processed food products has important application in halal authentication as well as in health, religions, and fare trades. The assay based on a pair of canine-specific primers that targeted a 100 bp region of canine mithochondrial-cytb gene which is present in multiple copies and highly conserved within the same species. The specificity of the assay was tested against dog and eight most common animal meat species as well as five plant species commonly found in frankfurter formulation. The stability and specificity of the assay were verified under different thermal processing conditions under pure and complex matrices. Three commercial brands of chicken and beef frankfurters were tested in triplicate, and specific PCR products were obtained only from deliberately contaminated formulations. The detection limit of the assay was 0.1 % (0.02 ng DNA) of canine meat spiked with other meats in a typical frankfurter formulation. Shorter amplicon length, superior stability, and higher sensitivity of the assay suggested its potential application in the screening of canine-origin biomaterials in processed food products.     

Determination of Sex Origin of Meat and Meat Products on the DNA Basis: A Review

Sex determination of domestic animal's meat is of potential value in meat authentication and quality control studies. Methods aiming at determining the sex origin of meat may be based either on the analysis of hormone or on the analysis of nucleic acids. At the present time, sex determination of meat and meat products based on hormone analysis employ gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography-mass spectrometry/mass spectrometry (HPLC-MS/MS), and enzyme-linked immunosorbent assay (ELISA). Most of the hormone-based methods proved to be highly specific and sensitive but were not performed on a regular basis for meat sexing due to the technical limitations or the expensive equipments required. On the other hand, the most common methodology to determine the sex of meat is unquestionably traditional polymerase chain reaction (PCR) that involves gel electrophoresis of DNA amplicons. This review is intended to provide an overview of the DNA-based methods for sex determination of meat and meat products.     

Application of the novel Droplet digital PCR technology for identification of meat species

The authenticity and traceability of meat products are issues of primary importance to ensure food safety. Unfortunately, food adulteration (e.g. the addition of inexpensive cuts to minced meat products) and mislabelling (e.g. the inclusion of meat from species other than those declared) happens frequently worldwide. The aim of this study was to apply a droplet digital PCR assay for the detection and quantification (copies μL⁻¹) of the beef, pork, horse, sheep, chicken and turkey in meat products. The analysis conducted on commercial meat showed the presence of traces of DNA from other animal species than those declared. We show that the method is highly sensitive, specific and accurate (accuracy = 100%). This method could be adopted by competent food safety authorities to verify compliance with the labelling of meat products and to ensure quality and safety throughout the meat supply chain, from primary production to consumption.     

Swine-Specific PCR-RFLP Assay Targeting Mitochondrial Cytochrome B Gene for Semiquantitative Detection of Pork in Commercial Meat Products

Verification of pork adulteration in commercial meat products is increasingly important for the authentication of Halal labels in processed foods. Here, we documented a PCR–restriction fragment length polymorphism (RFLP) assay with high precision and reproducibility for the tracing of porcine DNA in commercial meat products. The assay combined the species-specific primers to selectively amplify a short fragment (109 bp) of porcine cytochrome b gene from a heterogeneous background of genomic DNAs followed by RFLP analysis to authenticate real amplicon. The analysis of PCR products and restriction digests was automated in a chip-based capillary electrophoresis incorporated in Agilent 2100 bioanalyzer. The swine specificity of the assay was checked with 11 different meat-providing animal and fish species. Model experiments, mimicking the processed foods, were performed in binary and ternary mixtures after mechanical grinding and prolonged autoclaving. Finally, four types of the most popular finished meat products (meatball, streaky beacon, frankfurter, and burger) which are prevalent in the Malaysian food market were analyzed in order to verify the assay performance. The assay was sensitive enough to detect 0.0001 ng of swine DNA in pure formats and 0.01% (w/w) spiked pork in extensively processed ternary mixture of pork, beef, and wheat flour.